1. Field of the Invention
The present invention relates to a microorganism of the genus Eubacterium, which is suitable in pure culture or mixed culture for the detoxification of trichothecenes, and to a process for the isolation thereof, its production and formulation and its use and a feedstuff additive comprising the microorganism.
2. Description of the Prior Art
Trichothecenes which belong to the mycotoxins class are contained in numerous animal feedstuffs, where they are customarily introduced into the feedstuffs via mould fungi found on cereals or grasses. As a result of the undesired administration of mycotoxins, in particular trichothecenes, to animals, both their productivity and, for example, the growth of the animals is inhibited, an increased consumption of feedstuff together with a simultaneously poorer feedstuff utilization rate occurring in addition to damage to the health of the animals. To eliminate the adverse effects of mycotoxins, numerous processes for binding or adsorbing these toxins have already been disclosed.
Thus, in WO 91/13555, for example, a feedstuff additive and a process for the inactivation of mycotoxins is described, where particles of a phyllosilicate mineral are added to the feed in order to inactivate the mycotoxins. To increase the effect of these phyllosilicates, the particles are coated with a sequestering agent in order to accelerate the effect. A feedstuff is furthermore known, for example, from WO 92/05706 in which montmorillonite clay is contained as a feedstuff additive. These natural clay minerals having large internal surface areas should bind the mycotoxins to the surface on account of their porosity and immobilize them in this manner.
Furthermore, a feedstuff additive has been disclosed in the Austrian Utility Model AT-U 504 in which an enzyme preparation is used which is capable of forming epoxidases and lactonases and degrading mycotoxins chemically both in the feedstuff and in the gastro-intestinal tract of animals. According to AT-U 504, the action of this enzyme preparation can be increased by the addition of zeolites and the like.
The present invention now aims at making available a specific microorganism or a defined mixed culture isolated from a natural habitat, with which it is possible to convert mycotoxins, in particular trichothecenes, in a controlled manner into substances which are physiologically harmless and which are harmless, in particular in animal breeding, by biochemical degradation.
To solve this object, a microorganism of the genus Eubacterium was isolated which is suitable for the detoxification of trichothecenes in pure culture, DSM 11798, or mixed culture with the strain Enterococcus casseliflavus, DSM 11799, or other anaerobic microorganisms. According to a novel refinement of the invention, the microorganism is suitable for the detoxification of trichothecenes in mixed culture with other anaerobic microorganisms, in particular from the genus Enterococcus, Streptococcus, Lactococcus, Bacillus or Lactobacillus.
The microorganism of the genus Eubacterium, which is also called Eubacterium sp. on account of its association with the genus Eubacterium, and which was deposited in pure culture in the Collection of German Microorganisms under the number DSM 11798, or in mixed culture with the strain Enterococcus casseliflavus, which was deposited in the Collection of German Microorganisms under the number DSM 11799, is in particular suitable according to the invention for the detoxification of, in particular, deoxynivalenol (DON), T-2 toxin, HT-2 toxin, nivalenol, monoacetoxyscirpenol, diacetoxyscirpenol, trichodermol, verrucarin, rorodin, acetyldeoxynivalenol, isotrichodermin, hydroxyisotrichodermin, calonectrin, T-2 tetraol, T-2 triol, deacetylneosolaniol, neosolaniol; acetylneosolaniol, sporotrichiol, trichotriol, sambucinol and culmorin. The microorganism according to the invention detoxifies the trichothecenes by reductive biotransformation of the epoxide group contained in the molecule, which epoxide group is responsible for the toxicity of the mycotoxins, in particular trichothecenes. In the trichothecenes corresponding to the following formula, the degradation of the epoxide group is carried out by reductive cleavage of the toxic 12,13-epoxy ring: 
DSM 11798 and DSM 11799 were deposited with DSMZ-DEUTSCHE SAMMLUNG VON MIKROOGANISMEN UND ZELLKULTUREN GmbH, Mascheroder Weg 1b, D-38124 Braumschweig, Germany, on Sep. 17, 1997.
The morphology of the microorganism according to the invention shows preferably that it is an anaerobic gram-positive, rod-like, non-spore-forming bacterium, in particular 0.1 to 3 xcexcm long, which occurs both individually, in pairs or in long chains, in particular up to approximately 150 xcexcm. Phylogenetic analysis of the microorganism according to the invention has in particular shown a 16S RNA sequence, namely
The sequence data of the microorganism being compared with known 16S RNA gene sequences of representative microorganisms which are part of the domain of bacteria. This comparison analysis showed the greatest correspondence to bacteria of the genus Eubacterium. However, it was not possible to find any gene sequence corresponding sufficiently to a known microorganism, from which it results that the microorganism according to the invention is a microorganism within the genus Eubacterium which has still not been isolated and classified to date. Physiological investigations, such as, for example, fermentation spectra, reduction of nitrate to nitrite, also clearly showed the association with the genus Eubacterium.
A further object of the present invention is to make available a process for obtaining both a pure culture of the microorganism DSM 11798 and its mixed culture with the strain Enterococcus casseliflavus, DSM 11799, and other anaerobic microorganisms, an optimization of yield both in the economical and in the quantitative respect being aimed at in particular.
To achieve this object, the process according to the invention is carried out in such a way that a mixed culture DSM 11799 is obtained from the microorganism and Enterococcus casseliflavus from bovine rumen by culturing or fermenting at least twice in dilution series and anaerobic culturing conditions. To obtain both the mixed culture and the pure culture of the microorganism according to the invention, culturing and/or fermenting in dilution series at least twice has proved favourable, since in this manner an ensured purity of the desired products and, in particular, a removal of interfering by-products or contaminations with undesired microorganisms can be achieved. To maintain the anaerobic conditions, the culturing and/or fermentation according to the invention was preferably performed in a gas atmosphere of H2 and CO2, the gas atmosphere having a ratio of H2:CO2 of 10:90 to 90:10, in particular approximately 80:20, being particularly preferably selected. For the growth of the microorganism according to the invention, anaerobic conditions with a low redox potential are important, it surprisingly only being possible to achieve a sufficiently rapid growth in the presence of H2.
An even more rapid growth of the microorganism according to the invention can be achieved by carrying out the culturing and/or fermentation at an overpressure of 0.2 to 3 bar, in particular 0.5 to 1 bar, as this corresponds to a further preferred embodiment. It was possible to achieve further improved growth of the microorganism DSM 11798 according to the invention by preferably carrying out the culturing and/or fermentation at a temperature of 35 to 42xc2x0 C., in particular approximately 37xc2x0 C. The pH optimum for the culturing or fermentation in the process according to the invention was preferably a pH of between 6 and 8 and in particular between 7 and 7.5. Under these conditions, it is possible to obtain both a pure culture of the microorganism DSM 11798 and its mixed culture (DSM 11799) described above in as short a time as possible and using relatively few dilution series. Optimal results can be achieved with the process according to the invention by preferably carrying out the culturing and/or fermentation in a media preparation comprising components selected from: arginine, citrulline, peptone, yeast extract, fatty acid mixture(s), mineral solution(s), glucose, haemin solution, menadione, vitamin solution, trace elements and reducing agents.
The components contained in a media preparation are in this case partially exchangeable, it being possible, for example, by addition of glucose to achieve a shift of the equilibrium in the mixed culture in the direction of Enterococcus casseliflavus or corresponding other anaerobic microorganisms, it being possible to control the process specifically depending on the amount of glucose added. According to a particularly preferred aspect of the invention, at the start of the culturing and/or fermentation 0.1 to 0.5% by weight, in particular 0.2% by weight, of glucose is added. By addition of 0.1 to 0.5% by weight of glucose, the growth of Enterococcus casseliflavus is promoted at the start of the culturing and/or fermentation, which leads to a fall in the redox potential. By lowering the redox potential, optimum growth conditions for the microorganism according to the invention were created, so that, for example, chemicals, such as cysteine, in the media preparation can be dispensed with by means of a controlled addition of glucose.
In order to achieve the detoxification of mycotoxins, in particular trichothecenes, to other advantageous effect with the microorganisms and/or mixed culture according to the invention, enzyme preparation of the active, trichothecene-detoxifying microorganism and/or other anaerobic microorganisms can preferably also be added according to the invention.
To obtain a pure culture of the microorganism DSM 11798, the process according to the invention is preferably carried out such that the pure culture of the microorganism DMS 11798 is obtained from the culture or fermentation solution DSM 11799 by at least two further dilution series in the media preparation, in particular with addition of L-arginine as a growth stimulator. By carrying out two further dilution series in the media preparation from the culture or fermentation solution, the microorganism DMS 11798 can be obtained completely pure from the mixed culture with Enterococcus casseliflavus, the addition of the growth stimulator L-arginine advantageously shifting the equilibrium in the direction of the pure culture of the microorganism. In this connection, the growth of the bacterium according to the invention is promoted the higher the concentration of L-arginine.
In order to lower the redox potential of the media preparation further, a procedure is preferably used according to the invention in which, for the fermentation of the pure culture of the media preparation, 1 to 4% by weight of a reducing agent, in particular of a mixture of cysteine/sodium sulphide/sodium carbonate solution, is added. Particularly for reasons of economy, according to the invention the addition of the reducing agent is kept as low as possible, it having been shown in the course of comparison experiments that an increase in the concentration of the reducing agent beyond 4% by weight causes no further acceleration of the growth of the microorganism.
To obtain a storable finished product, the process according to the invention is preferably continued by working up the culture or fermentation solution by concentrating, in particular centrifuging or filtering and/or stabilization, in particular by freeze- or spray-drying or encapsulating. In this connection, for example, the culture or fermentation solution is concentrated in a first step by removing liquid by centrifuging or filtering, and/or carrying out the stabilization directly from the fermentation solution, preferably with addition of a filler or carrier material, such as aluminium silicates, kieselguhrs, carbohydrates, sugar alcohols, starches, milk and whey powder, protein hydrolysates, yeasts and PVPP. By addition of these carriers or fillers, it is possible in the following stabilization step, in particular the So freeze-drying, spray-drying, encapsulation of pelletization step, to obtain a solid product in which the pure culture of the microorganism DSM 11798 or its mixed culture with the strain Enterococcus casseliflavus, DSM 11799, or other anaerobic microorganisms, in particular from the genus Enterococcus, Streptococcus, Lactococcus, Bacillus or Lactobacillus, which are suitable for the detoxification of trichothecenes, is deposited directly on a carrier, as a result of which a particularly easily manageable and storable as well as metabolically favourable product is obtained. By depositing the microorganism or its mixed culture on a substance having a large internal surface area, such as argillaceous earths, aluminium silicates, zeolites and the like, the intended degradation according to the invention of trichothecenes is further facilitated, since these are bound physically to the substance having a large internal surface area, whereupon the biochemical attack with the microorganism according to the invention is distinctly facilitated.
According to the invention, the microorganism is further used in pure and/or mixed culture (DSM 11798, DSM 11799) for the production of a feedstuff additive. A particularly preferred use according to the invention essentially results in that the pure and/or mixed culture (DSM 11798 and DSM 11799) is employed as a freeze- or spray-dried and/or encapsulated or pelleted immobilizate, if appropriate with addition of a carrier material. Both the pure and/or mixed culture of the microorganism according to the invention and the spray-dried immobilizate can be used directly as a feedstuff additive, it even being possible to admix the feedstuff additive to the feedstuff directly during preparation and/or to mix it into the feedstuff either in solid or in liquid form during feeding to the animals.
In order to achieve an as complete as possible degradation or chemical conversion of the trichothecenes into physiologically acceptable substances, a feedstuff additive according to the invention for the inactivation of trichothecenes in feedstuffs or in the digestive tract of animals is essentially characterized in that the feedstuff additive contains a pure and/or mixed culture of a microorganism according to the invention or of a microorganism prepared according to the invention in an amount from 105 to 1012 cells/kg, in particular 107 to 109 cells/kg, per 1000 kg of feedstuff. By use of 105 to 1012 cells/kg, in particular 107 to 109 cells/kg, of the microorganism according to the invention or of the mixed culture according to the invention of the microorganism and Enterococcus casseliflavus or other anaerobic microorganisms, in particular of the genus Enterococcus, Streptococcus, Lactococcus, Bacillus or Lactobacillus, which are suitable for the detoxification of trichothecenes, it is possible to convert high concentrations of trichothecenes, in particular of deoxynivalenol, T-2 toxin, HT-2 toxin, nivalenol, monoacetoxyscirpenol, diacetoxyscirpenol, trichodermol, verrucarin, rorodin, acetyldeoxy-nivalenol, isotrichodermin, hydroxyisotrichodermin, calonectrin, T-2 tetraol, T-2 triol, deacetyl-neosolaniol, neosolaniol, acetylneosolaniol, sporo-trichiol, trichotriol, sambucinol and culmorin in feedstuffs into chemically harmless substances, such as the deepoxy metabolite of deoxynivalenol (DOM-1), so that, using the feedstuff additive according to the invention, both an increase in productivity of the animals and, on account of the reduced toxicity, an improved feedstuff conversion rate can be achieved.
In order to further facilitate the biochemical degradation of the mycotoxins, in particular trichothecenes, according to the invention a carrier material and/or filler can preferably be additionally contained in the feedstuff additive in an amount of 0.5 to 8 kg/1000 kg, in particular 0.7 to 4 kg/1000 kg, of the feedstuff. By means of the addition of carrier materials and/or fillers, it is possible, if desired, to bind the mycotoxins and also other harmful substances to be degraded which can be contained in the feedstuff, physically to the substances, as a result of which they are no longer available for metabolization.
In this case, in particular, aluminium silicates, kieselguhrs, carbohydrates, sugar alcohols, starch, milk and whey powder, protein hydrolysates, yeasts and/or PVPP are employed as a carrier material and/or filler, these carrier materials and/or fillers having proved to be particularly advantageous for the binding of toxins, in particular trichotoxins.
A particularly preferred feedstuff additive is characterized in that the feedstuff additive consists of a mixture of 1 to 65% by weight, in particular 5 to 50% by weight, of the spray- or freeze-dried immobilizate of the microorganism and 99 to 35% by weight, in particular 95 to 50% by weight, of carrier material and/or filler. Feedstuff additives of this type are suitable, in particular, for the inactivation of deoxy-nivalenol (DON), T-2 toxin, HT-2 toxin, nivalenol, monoacetoxyscirpenol, diacetoxyscirpenol, trichodermol, verrucarin, rorodin, acetyldeoxy-nivalenol, isotrichodermin, hydroxyisotrichodermin, calonectrin, T-2 tetraol, T-2 triol, deacetyl-neosolaniol, neosolaniol, acetylneosolaniol, sporo-trichiol, trichotriol, sambucinol and culmorin both in the feedstuff and in the digestive tract of animals.